Practical 1 - Investigation into size and scale of microscopic tissues

This practical focuses on microscope technique and using graticules and stage micrometres to
determine size and scale in biological cells and tissues.

Intended learning outcomes

By the end of this practical you should be able to:

 Use a microscope fitted with an eyepiece graticule and stage micrometer

 Calibrate the eyepiece graticule using the stage micrometer

 Use the calibrated graticule to determine the actual size of microscopic

 specimens

 Estimate the accuracy of a measurement

 Use the graticule to determine scales

 Understand the importance of repeating or validating set of results.

Safety Information

There are no particular hazards in this practical, however you must follow your laboratory rules.

Background information

The measurement of specimen size with a microscope, is made by using an eyepiece graticule. This is
a glass or plastic disc with 8 divisions etched onto its surface, which is inserted into the eyepiece
lens.

The size of the eyepiece graticule remains constant, despite the fact that the image viewed will
change its size depending upon whether high- or low-power objective lenses are used. For example a
cell viewed with the x40 objective will appear much larger than when viewed with the x10 objective.
However because the graticule is in the eyepiece it will not change its size. Therefore the value of
each of the divisions in the eyepiece graticule varies with the magnification of the objective lens.

A stage micrometer is a very accurately etched glass or plastic ruler that is placed on the microscope
stage so that the eyepiece graticule scale is superimposed on the stage micrometer scale. In this lab
we will be using a 10 mm or 1 cm scale micrometer divided into 10 large divisions. Each division is
then divided into 10 additional divisions, making a total of 100 small divisions.
1 stage micrometer length = 1cm = 10 mm.

There are 100 divisions (10 x 10) on the micrometer.

Therefore the smallest division on the micrometer = its length/total number of divisions = 1cm/ 100
= 0.01cm

or 10 mm/ 100 = 0.1 mm

It is necessary to calibrate the eyepiece graticule with the stage micrometer placed on the
microscope stage for each objective lens used. You will observe aTS (transverse section) of plant
tissues through a microscope and use an eyepiece graticule and a stage micrometer to determine
the size of some of the structures.

Read the information above. Ensure that you understand the principles of using an eyepiece
graticule and a stage micrometer before you continue with the investigation.

Method

Preparation

1. You have been provided with a compound light microscope with both low and high-power
objective lenses and an eyepiece lens that has been fitted with a graticule. You have also been
provided with a stage micrometer.

Label the microscope: objective lens, stage, light source, eye piece.

2. You must now calibrate the eyepiece graticule.

Place the stage micrometer onto the microscope stage and focus using the low-power objective lens
so that the graticule scale becomes superimposed over the stage micrometer scale.

3. Move the stage micrometer until the start or zero line of each scale is coincident (lined up)

4. Look along the scale until another coincident point is found.

 5. The relationship between the two scales can now be
calculated. On the scale shown there are 25 small divisions on
the stage micrometer scale that line up with 100 small divisions
on the graticule scale.

Thus 1 graticule division = 25 / 100 = 0.25 stage micrometer

units.

Each unit on the stage micrometer scale is 0.1 mm = 100

micrometres (100μm). Therefore each division on the graticule
scale is 0.25 x 0.1mm = 0.025 mm to 25 μm.

6. Use the procedure described above to determine the size of each division on the eyepiece
graticule using the low-power objective lens of your microscope.

7. Repeat the procedure to determine the size of each division when using the high-power objective
lens.

Making observations

1. You are provided with a stained transverse section through part of a dicotyledonous plant root.
2. Examine the specimen using the low-power of your microscope.

3. Make a large, plan drawing to show the distribution of tissues, labelling the stele (vascular
bundle).

4. Use the eyepiece graticule to measure the width of the vascular bundle at its widest point in
graticule units and then calculate the actual width of the vascular bundle in millimetres and in
micrometres.

5. Draw a straight line on your drawing across the vascular bundle to show where you took your
measurement. Write the dimension on your drawing next to the line.

6. Make a high-power drawing to show a group of four xylem vessels from inside the vascular
bundle.

7. Use the eyepiece graticule to measure the width of the xylem vessel at its widest point in graticule
units and then calculate the actual width of the vessel in micrometres, remembering to use the
appropriate calibration of the eyepiece graticule for the high-power objective lens.

8. Draw a straight line on your drawing across the xylem vessel to show where you took your
measurement. Write the dimension on your drawing next to the line.

9. Look at your two measurements and check on their accuracy. The actual size of the xylem vessel
should be smaller than the size of the vascular bundle even though it looked larger using the high
power objective lens.

10. You are now going to determine the magnification of your drawing of the xylem vessels. Use a
ruler to measure the length of the line that you drew across the xylem vessel.

Use your knowledge of the actual size of the vessel to calculate the magnification of your drawing.
Write your answer x at the bottom right hand corner of your drawing.

Follow-up

1. Compare your results with other members of the class and check for consistency of readings.
 2. Did any member of the class have anomalous results? What are the potential causes of such
an anomalous result in this investigation?

3. Write up your procedure including a discussion of the benefits of comparing your results
with other students.

Note: 1 micro-meter = 1.0 × 10-6 metres = 1.0 x 10 -3 mm

1 metre = 1,000,000 = 1 x 106 micro-metres =

1 metre = 1 x109 nano-metres

Sample Calculation (You have already determined that 1 small division of the stage micrometer =
0.1mm)

You measure the length of an onion cell under the microscope and it measures 23 small divisions of
the eyepiece graticule. When you measure the graticule against the stage micrometer, 3 small
graticule divisions = 1 small division of the stage micrometer. What is the length of the onion cell?

3 graticule divisions = 1 stage micrometre divisions which is 0.1 mm

Length of 1 graticule division = 0.1 mm/3 = 0.033 mm

The onion cell is 23 graticule divisions

Length of onion cell = 23 x 0.033mm = 0.759 m = 0.76 mm